This invention relates generally to the production, maintenance and control of transgenes in transgenic eukaryotic organsims that undergo meiosis in which pollen or sperm can be outcrossed; this includes: transgenic animals, plant cells, plant tissues and whole plants. More specifically, this invention relates to the control of transgene transmission by male and/or female gametes or gametophytes. The genetic constructs and methodologies of the present invention provide the ability to control the undesired spread of transgenes. In addition, this invention also provides the tools and methodologies to enrich a plant genome, or any other eukaryotic genome, for dispersed and/or stable transposition events.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Transgenic crops and the application of biotechnology are dramatically altering seed and agrochemical businesses throughout the world. The seeds of commercially important crops have been genetically engineered to be resistant to herbicides and pests, especially insect pests. According to surveys by the United States Department of Agriculture (June, 2000), genetically modified corn, soybeans and cotton were grown on approximately 25%, 54% and 61%, respectively, of the total U.S. acres for each crop in 2000.
The uncontrolled transmission of heterologous traits in commercially important crop plants is currently a major concern throughout the world and especially within the agricultural community. The undesired dissemination of transgenic pollen may unintentionally harm beneficial insects and may result in the spread of transgenes to related plant species leading to the contamination of food products and the production of herbicide- and pesticide-resistant weedy species.
The biotechnology industry is interested in transferring traits such as tolerances to drought, insects, diseases, salinity, frost and herbicides into cultivated plants which might confer an adaptive advantage over wild plants. Several crop species are known to be cross-compatible with wild species and it is possible that these traits could be inadvertently transferred to wild weedy relatives through sexual hybridization leading to possible economic and ecological harm. Since most forage and turf grasses have undergone relatively little domestication and may even be considered weeds in certain instances (e.g., bermudagrass), there is a high probability of greater problems in the ultimate release and use of such genetically transformed plants. Because forage grasses, in general, are not highly domesticated, possess weedy characteristics, and are highly outcrossing, special difficulties may be encountered in the ultimate release of transgenic forage grasses.
It would be highly desirable to have a method to prevent the undesired transmission of heterologous traits in commercially important crop plants. If this could be achieved, genetic leakage of heterologous traits would be brought under control and the spread of these traits to undesired recipients would be curtailed. Thus, the need exits for a genetic system that selects against male or female gametophytes containing transgenes, thereby preventing, eliminating or reducing the undesired transmission of heterologous traits. In particular, there is a need for a genetic system which allows for the transmission of non-transgenic (i.e., wild type) gametophytes while preventing the transmission of the transgenic (i.e., heterologous) gametophytes from the same plant.
Thus, an object of this invention is to provide recombinant nucleic acid constructs and methods for controlling, reducing or eliminating the undesired transmission of heterologous traits in commercially important crop plants.
The instant invention is directed to genetic constructs and methods for controlling the spread of heterologous traits in plants. Control is achieved by providing a sex-specific promoter operably linked to a suicide gene that selects against male or female gametes containing the suicide gene. The suicide gene locus is termed the xe2x80x9cgametophytic suicide traitxe2x80x9d (GST) (FIG. 1A). By linking a transgene of interest to a suicide gene under the control of a sex-specific promoter, transmission of the transgene to progeny is effectively eliminated, reduced or prevented because no gametes bearing the GST will be produced.
In one aspect, the invention can be said to broadly consist of a suicide gene under the control of a pollen-specific promoter linked to a transgene of interest. The transgene complex can be introduced into a virgin plant genome and plants can be selected which are hemizygous for the transgene complex. The only pollen produced by the hemizygous plant will lack the transgene complex due to its physical linkage to the suicide gene. Uncontrolled spread of the heterologous trait encoded by the transgene complex is thereby prevented because no pollen containing the transgene complex is produced.
A second aspect of the invention is based on placing the GST in close proximity to a transposon to produce selective enrichment of dispersed transposition events in progeny cells since only those gametes lacking the GST locus will be viable. Since a fraction of the progeny cells produced from viable gametes will have undergone transposition events, selective enrichment of dispersed transposition events is achieved because the transposon is necessarily no longer linked to the GST (the GST destroys those gametes that inherit the GST gene locus) (FIG. 1B).
Thus, the present invention provides genetic systems which can be used for the elimination of a GST transgene complex and for the selection for unlinked transpositions. By using the GST together with any transgene, one can completely eliminate male (or female in the case of a female gametophytic-specific promoter:suicide construct) transmission of both the GST and the associated transgene (FIG. 2).
This invention provides nucleic acid constructs comprising a male gamete- or female gamete-specific promoter operably linked to a suicide gene, wherein said promoter and said suicide gene combination is linked to a gene of interest.
This invention provides nucleic acid constructs comprising a male gamete- or female gamete-specific promoter operably linked to a suicide gene, wherein the promoter and the suicide gene combination is linked to a gene of interest. This invention further provides such nucleic acid constructs wherein the promoter is selected from the group consisting of a pollen-specific promoter and an ovule-specific promoter. This invention still further provides such nucleic acid constructs wherein the suicide gene is selected from the group consisting of barnase, tasselseed2 and diphtheria toxin A gene. This invention also provides such nucleic acid constructs wherein the gene of interest is selected from the group consisting of a nucleic acid encoding herbicide resistance, antibiotic resistance, insecticide resistance, nitrogen fixation, improved nutrition and cellulose content.
This invention provides nucleic acid constructs comprising a pollen-specific promoter or an ovule-specific promoter operably linked to a suicide gene selected from the group consisting of barnase, tasselseed2 and diphtheria toxin A gene; wherein the promoter and the suicide gene combination is linked to a gene of interest selected from the group consisting of a gene coding for herbicide resistance, antibiotic resistance, insecticide resistance, nitrogen fixation, improved nutrition and cellulose content or other agronomic trait of interest.
This invention provides methods for reducing or eliminating male transmission of a transgene locus in a plant comprising:
a) transforming a plant cell with a nucleic acid construct in which a male gamete-specific promoter is operably linked to a suicide gene, wherein said promoter and said suicide gene combination is linked to a heterologous polynucleotide;
b) propagating said transformed plant cell through meiosis to produce male gametes lacking said transgene locus.
This invention also provides methods for reducing or eliminating male transmission of a transgene locus in a plant comprising:
a) transforming a plant cell with a nucleic acid construct in which a pollen-specific promoter is operably linked to a suicide gene;
i) wherein said suicide gene is selected from the group consisting of bamase, tasselseed2 and diphtheria toxin A gene;
ii) wherein said promoter and said suicide gene combination is linked to a heterologous polynucleotide;
iii) wherein said heterologous polynucleotide is selected from the group consisting of DNA encoding herbicide resistance, antibiotic resistance, insecticide resistance, nitrogen fixation, improved nutrition and cellulose content;
b) propagating said transformed plant cell through meiosis to produce male gametes lacking said transgene locus.
This invention also provides methods for reducing or eliminating female transmission of a transgene locus in a plant comprising:
a) transforming a plant cell with a nucleic acid construct in which a female gamete-specific promoter is operably linked to a suicide gene, wherein said promoter and said suicide gene combination is linked to a heterologous polynucleotide;
b) propagating said transformed plant cell through meiosis to produce female gametes lacking said transgene locus.
This invention further provides methods for reducing or eliminating female transmission of a transgene locus in a plant comprising:
a) transforming a plant cell with a nucleic acid construct in which an ovule-specific promoter is operably linked to a suicide gene;
i) wherein said suicide gene is selected from the group consisting of barnase, tasselseed2 and diphtheria toxin A gene;
ii) wherein said promoter and said suicide gene combination is linked to a heterologous polynucleotide;
iii) wherein said heterologous polynucleotide is selected from the group consisting of DNA encoding herbicide resistance, antibiotic resistance, insecticide resistance, nitrogen fixation, improved nutrition and cellulose content.
b) propagating said transformed plant cell through meiosis to produce female gametes lacking said transgene locus.
This invention also provides transformed plant cells produced by the methods of the present invention wherein the transformed plant cells are hemizygotic for the nucleic acid construct.
This invention provides nucleic acid constructs comprising a male gamete- or female gamete-specific promoter operably linked to a suicide gene wherein said promoter and said suicide gene combination is linked to a transposable element. This invention also provides such nucleic acid constructs which further comprise one or more transposase genes. This invention further provides such nucleic acid constructs which further comprise one or more genes of interest. This invention still further provides such nucleic acid constructs wherein the gene of interest is associated with the transposable element.
This invention provides nucleic acid constructs in which a pollen-specific promoter or an ovule-specific promoter is operably linked to a suicide gene selected from the group consisting of bamase, tasselseed2 and diphtheria toxin A gene; wherein said promoter and said suicide gene combination is linked to a transposon, wherein said transposon comprises a selectable marker selected from the group consisting of a gene coding for herbicide resistance, antibiotic resistance, insecticide resistance, nitrogen fixation, improved nutrition and cellulose content. This invention also provides such nucleic acid constructs wherein the promoter is selected from the group consisting of a pollen-specific promoter and an ovule-specific promoter. This invention also provides such nucleic acid constructs wherein the suicide gene is selected from the group consisting of bamase, tasselseed2 and diphtheria toxin A gene.
The present invention provides methods for enriching dispersed transposition events in a population of plant cell progeny comprising:
a) transforming a plant cell with the nucleic acid construct of any one of the aforementioned nucleic acid constructs to produce a transformed plant cell;
b) propagating said transformed plant cell through meiosis to produce plant cell progeny in which dispersed transposition events are enriched.
The present invention also provides such methods which include the additional step of isolating the plant cell progeny in which dispersed transposition events are enriched. The present invention also provides plant cells and plants which contain dispersed transposition events and, particularly, the plant cells and plants are hemizygotic for the nucleic acid.
The present invention provides nucleic acid constructs comprising a first promoter wherein the first promoter is a male gamete- or female gamete-specific promoter operably linked to a suicide gene and further comprising a nucleic acid encoding a transposase and a nucleic acid encoding a transposon. The present invention also provides such nucleic acid constructs wherein the transposon comprises a second promoter operably linked to a selectable marker, wherein the selectable marker is not a suicide gene.
The present invention provides nucleic acid constructs in which a pollen-specific promoter or an ovule-specific promoter is operably linked to a suicide gene selected from the group consisting of bamase, tasselseed2 and diphtheria toxin A gene, wherein said promoter and said suicide gene combination is linked to a nucleic acid encoding transposase; wherein said promoter and said suicide gene combination linked to said nucleic acid encoding transposase comprise a transgene locus which further comprises a transposon; wherein said transposon comprises a polynucleotide sequence encoding a member selected from the group consisting of herbicide resistance, antibiotic resistance, insecticide resistance, nitrogen fixation, improved nutrition and cellulose content. The present invention provides such nucleic acid constructs wherein the promoter is selected from the group consisting of a pollen-specific promoter and an ovule-specific promoter. The present invention further provides such nucleic acid constructs wherein the suicide gene is selected from the group consisting of bamase, tasselseed2 and diphtheria toxin A gene. The present invention also provides such nucleic acid constructs wherein the transposon comprises a polynucleotide sequence encoding a member selected from the group consisting of herbicide resistance, antibiotic resistance, insecticide resistance, nitrogen fixation, improved nutrition and cellulose content.
The present invention also provides methods for enriching stably dispersed transposition events in a population of plant cell progeny comprising:
a) transforming a plant cell with a nucleic acid constructs of the present invention to produce a transformed plant cell;
b) propagating said transformed plant cell through meiosis to produce plant cell progeny in which stably dispersed transposition events are enriched.
The present invention also provide such methods further comprising the step of isolating the plant cell progeny in which the stably dispersed transposition events are enriched. The invention further provides plant cells isolated by such methods and plants produced from the plant cells.
This invention provides vectors comprising the nucleic acid constructs of the present invention as well as host cells, recombinant plant cells and transgenic plants comprising the vectors of the present invention. More particularly, this invention provides such cells and transgenic plants which are hemizygotic for the nucleic acid constructs.